Specific Aims. Apolipoprotein A-I (apo-A-I) is the main protein component of high density lipoprotein (HDL) and acts as an activator of the enzyme lecithin cholesterol acyltransferase (LCAT). The long term objective of this proposal is to understand the structure-function relationships of human apoA-I. In this application emphasis will be placed on the domains and residues on the protein which 1) participate in the activation of LCAT, and 2) are important in stabilizing the conformation of A-I both in solution and in association with lipid. It is our hypothesis, based on existing experimental data and models, that the ability of apoA-I to associate with lipid in the form of discoidal phospholipid bilayer complexes or spherical HDL and activate LCAT depends on the three- dimensional arrangement of the amphipathic helical repeats. The specific aims are: 1. To mutagenize the human apo-A-I gene, generate by transfection and selection cell lines which express variant forms of apoA-I, and isolate the proteins from the culture media for structural and functional analysis using established methodologies. The selection of the mutations will be based on three criteria: i) The importance of certain apoA-I domains for LCAT activation and lipid binding as suggested by experimental data and existing models. ii) The preference of charged amino acids for topologically specific positions which may play a role in electrostatic interaction that stabilize the side-by-side interactions of a-helices in apoA-I both in solution and in association with lipids; iii) the location of leucine zipper motifs and other residues in specific positions in the a-helices, which may stabilize the tertiary folding of A-I in solution in a helical bundle or may be involved in the association with lipids. 2. To study the thermodynamic stability of these variants forms of apoA-I with a view to establishing the molecular details important for the maintenance of the secondary and tertiary conformation required for the functional roles of apoA-I both in solution and in association with lipids. 3. To study the functional properties of the mutants apoA-I forms particularly their ability to activate LCAT and to bind to phospholipid cholesterol vesicles, model lipid emulsions and to HDL. Changes in LCAT activation and lipid binding will then be correlated with predicted or observed alterations in the apoA-I structure. Human subjects with low or high plasma apoA-I or HDL have an increased or decreased risk respectively of developing atherosclerosis. Understanding the structural elements of apoA-I which determine its secondary and tertiary conformation is essential to understand the function and anti-atherogenic properties of this very important protein.